Amino acid compositions of thiolases (seleno-thiolase and non-selenothiolase) were found to be similar. The native enzyme 158,000 to 160,000 Mr contains 16 cysteine residues but only 4 of these were titratable with DTNB or were detected as (14-C)carboxymethyl cysteine in hydrolysates of native protein alkylated with (14-C)iodoacetic acid. (75-Se)thiolase isolated from Clostridium kluyveri contained selenium in the form of (75-Se)selenomethionine. This seleno amino acid was identified by chromatography on an amino acid analyzer, by TLC analysis and by its ability to be converted to a product indistinguishable from Se-adenosylseleno-methionine by S-adenosyl methionine synthetase. Inhibition of growth of Clostridium sticklandii by the antibiotic monensin also resulted in inhibition of the synthesis of (75-Se)selenoprotein A. Radioactivity that initially copurified with the tRNA fraction from the monensin-treated cells was unstable suggesting that a precusor to the seleno-tRNAs normally formed had accumulated in the presence of monensin.